Our long-term goal is to develop a sensitive, multiplexed detection platform for real-time single-cell monitoring of prognostic kinase activity in tumor samples. The objective of this application is to develop the first steps towards a multiplex quantification of kinase activity in a breast cancer model system using surface-enhanced Raman spectroscopy (SERS) and peptide-functionalized nanoparticle (NP)-based biosensors. Recent unpublished work in our laboratory suggests that exogenously-added peptide substrates and SERS will allow for sensitive, direct monitoring of kinase activity in biological environments such as cells. In this multidisciplinary application, we combine the kinase biosensor expertise of Parker lab and the extensive SERS experience of Irudayaraj lab to develop a quantitative SERS platform to monitor the activity of Akt, Erk, Src, and c-Abl, kinases associated with drug resistance and clinical outcome of breast cancer patients. Towards demonstrating this proof-of-concept, we propose the following specific aims: Specific Aim 1. Standardize a SERS platform for the quantification of Akt, Erk, Src and Abl kinase activity. Specific Aim 2. Develop single cell mapping schemes to monitor differential kinase activity in response to various stimuli and inhibitors. Our approach represents a novel use of SERS microscopy and kinase substrate NP biosensor design. This technology is non-destructive (leaving cells viable and intact after analysis) and is adaptable to single molecule (and thus single cell) microscopy formats, providing exquisite spatial resolution and allowing us to monitor localized signaling in living cells. It is also tunable for different kinase substrates to allow simultaneous monitoring of more than one kinase activity, in other words 'multiplexing' the analysis, so we will be able to visualize kinases as complexes and systems rather than in isolation. This project has the potential to transform personalized medicine and therapy selection by facilitating single cell monitoring of therapeutic response. Using handheld SERS devices in the clinic, this technology could potentially benefit thousands of patients by giving pathologists (and thus clinicians) nearly 'real-time' mechanistic information about an individual's disease and therapeutic response. This work will also advance the field of signal transduction biology as a whole by enabling the analysis of multiple kinase activities (as opposed to just detecting the phosphorylation state of known endogenous substrates) in single cells in a microscopic platform. Upon demonstrating proof-of- concept with this pilot project, we will have the tools and experience in hand to undertake further technology and instrumentation development to facilitate real-time, live cell imaging using our technique, as well as implementation of a point-of-care, handheld SERS readout using commercially-available devices (goals for a future R33 application). Maturation of the detection platform to that stage would allow biologists for the first time ever to detect signal transduction in real-time in live cells without needing to develop and express e.g. a fluorescent genetic construct, potentially transforming cancer research and drug discovery.